Exploring genetic variation in Gossypium arboreum L. germplasm is useful as it contains many important genes conferring resistance to different stresses. In limited earlier studies, low level of genetic diversity was found by using conventional DNA marker systems which may impede future genome mapping studies. In the present investigation, we explored the extent of Single Nucleotide Polymorphisms (SNP) among 30 conserved regions of Expressed Sequence Tags (EST) of low copy genes between two genotypes of G. arboreum. A total of 27 SNPs including 21 substitutions and 6 Insertions and deletions (Indels) in 7804 bp were found between these genotypes with a frequency of one SNP per 371 bp and one Indel after every 1300 bp. Out of these SNPs, 52% were transitions, whilst 48% SNPs were transversion. In conclusion, SNPs are expedient markers that can explore polymorphism in highly conserved sequences where other markers are not effective.